Download introduction methods results and discussion conclusions

Prediction of Cholestatic Hepatotoxicity: Role of Transporter Regulation and Adaptive Response
Jonathan P. Jackson, Kimberly M. Freeman, Robert L. St. Claire III, and Kenneth R. Brouwer
Qualyst Transporter Solutions, 2810 Meridian Parkway, Suite 100, Durham, NC 27713
INTRODUCTION
Impaired canalicular efflux of bile acids has been postulated to
play a role in drug-induced liver injury (DILI). Cholestatic DILI
potential in humans has been associated with bile salt export
pump (BSEP) inhibition; however, in vitro BSEP inhibition potency
determinations do not correlate with in vivo cholestatic DILI
severity. In vivo concentrations of bile acids are tightly regulated
through synthesis, metabolism and transport mechanisms. Past
studies have focused on acute interactions without evaluating the
potential for adaptive responses (chronic interactions) of the
hepatocyte. Chenodeoxycholic acid (CDCA) was used as a
model bile acid (BA) to evaluate the concentration (72 hour
exposure) and time course effects of chronic BA exposure on BA
disposition in Transporter Certified™ sandwich-cultured human
hepatocytes (SCHH). B-CLEAR® technology was used to assess
the hepatobiliary disposition of d8-TCA, d5-GCDCA (endogenously
generated), and the intracellular total bile acid (endogenously
generated) pool in SCHH following 1, 3, 6, 12, 24, 48 and 72
hours exposure to 100 µM CDCA or d5-CDCA. The mRNA
content of key regulatory factors, synthetic enzymes, and
transport proteins for BA was determined.
RESULTS AND DISCUSSION
Figure 4.
Normal bile acid
levels
Increased bile
acid levels
trigger adaptive
response
Figure 1. Intracellular concentration of d8-TCA (exogenous) and d5GCDCA (endogenously generated) following timed continuous
exposures to 100 µM CDCA or d5-CDCA.
Figure 2. Total endogenous bile acid pool (CA, CDCA, TCA, GCA,
TCDCA, GCDCA) over time following incubation with 0.1% DMSO
or 100 µM d5-CDCA.
Adaptive response: Induction of OSTα/β
and BSEP along with suppression of
CYP7A1 – leads to an increase in
basolateral and canalicular efflux, and
decreased intracellular concentration to
prevent of cholestatic hepatotoxicity
METHODS
Human Hepatocytes
Cryopreserved, Transporter
Certified™ human hepatocytes in a sandwich configuration (24well format) were cultured using Qual-Gro™ Induction Media;
experiments were performed on Day 5 of culture.
Treatments Hepatocyte cultures were exposed to 100 µM
CDCA or d5-CDCA in culture media continuously for 1, 3, 6, 12,
24, 48 or 72 hours. Treatment with DMSO (0.1%) was used as a
control.
Gene Expression mRNA content of various transporters,
synthetic enzymes, and regulatory factors from SCHH was
determined from each RT reaction using gene-specific TaqMan®
primer/probe sets. All reactions were normalized to the
endogenous control GAPDH. Amplifications were performed on
an ABI ViiA7 Real-Time PCR System in relative quantification
mode. Relative-fold mRNA content was determined for each
treatment group relative to the 0.1% DMSO vehicle control.
Endogenous Bile Acids LC-MS/MS which employed
reversed-phase HPLC and electrospray ionization was used to
quantitate endogenously generated cholic acid (CA), CDCA, and
their taurine (TCA, TCDCA) and glycine (GCA, GCDCA)
conjugates in cells, bile and cell culture media.
B-CLEAR® technology is covered by US Pat. No. 6,780,580 and other US
and International patents both issued and pending.
Figure 3. Changes in mRNA expression for
selected transporters, synthetic enzymes,
and regulatory factors following timed
continuous exposure to 100 µM CDCA. No
change (</> 2 fold of control) was observed
for BCRP, CYP8B1, MRP2, MRP3, MRP4,
NTCP, OATP1B3, OATP2B1, and PGP.
CONCLUSIONS
• Adaptive responses to increased intracellular concentrations of bile acids occurred as early as 3 hours,
•
•
•
•
and were maximal by approximately 24 hours.
In the presence of 100 µM CDCA, the ICC of both d8-TCA and d5-GCDCA were significantly reduced at
each exposure time, to 10.7% of control and 14.4% of control, respectively, after 72 hours of exposure
(Figure 1).
The ICC of the total bile acid pool was significantly reduced to < 10% of solvent control following
exposure to CDCA (Figure 2).
Exposure to CDCA decreased the CYP7A1 mRNA, and increased the mRNA content of BSEP and
OSTα/β (Figure 3).
The adaptive response (decreased synthesis and increased bile acid efflux by OSTα/β and BSEP),
decreased the intracellular concentration of bile acids which decreases the potential for hepatotoxicity
(Figure 4).
Contact information: [email protected]
919-593-2519 (cell); 919-313-0163 (fax)